Interleukin-6 (IL-6) is a term given to a protein described by numerous synonyms in the literature. Some examples include interferon-beta-2 (IFN-B2), B-cell stimulation factor-2 (BSF-2), B-cell hybridoma/plasmacytoma growth factor (HPGF or HGF), 26 kDa protein and hepatocyte stimulating factor (HSF); see interferon-beta-2 (Zilberstein et al., EMBO J. 5, 2519 (1986)); 26 kDa protein (Haegeman et al., Eur. J. Biochem. 159, 625 (1986)); B-cell stimulation factor-2 (Hirano et al., Nature 324, 73 (1986)); B-cell hybridoma/plasmacytoma growth factor (Van Snick et al., Proc. Natl. Acad. Sci. 83, 9679 (1986)); Billiau, Immunol. Today 8, 84 (1987)); Van Damme et al., Eur. J. Biochem. 168, 543 (1987)); Tosato et al., Science 239, 502 (1988); and hepatocyte stimulating factor (Gauldie et al., Proc. Natl. Acad. Sci. 84, 7251 (1987)).
The sequence of hybridoma growth factor, and, therefore, of IL-6, is given by Brakenhoff et al, Journal of Immunology 139, 4116-4121 (1987); see FIG. 2A on page 4119. The amino acid sequence consists of a signal peptide followed by the mature, full-length HGF protein.
There is controversy regarding the position at which the signal peptide ends and the mature protein begins. The signal peptide is described as ending with the 27th amino acid (proline) or the 28th amino acid (alanine). The mature full-length protein contains the 185 amino acid residues starting with alanine or the 184 amino acid residues starting with proline. Different sources produce different mixtures of full length and truncated forms of IL-6, including full length IL-6, IL-6 lacking the first two amino acids, i.e. AlaPro and/or IL-6 lacking the first amino acid, i.e. Ala. See Van Damme, "Biochemical and Biological Properties of Human HPGF/IL-6" in Interleukin-6, Sehgal et al., eds., (Volume 557 of the New York Academy of Sciences), Page 104-109 at 107 (1989).
FIG. 2C in the Brakenhoff et al. article describes the differences in the nucleotide sequences that code for HGF, IFN-beta-2, 26 kd protein and BSF-2. These differences consist of a T at nucleotide 46 in 26 kd protein and a C corresponding to this position in the other three sequences; and a C at position 429 in IFN beta-2 and a G corresponding to this in this position in the other three sequences; see page 4118, column 1, the second full paragraph of Brakenhoff et al., Journal of Immunology 139, 4116-4121 (1987). Both differences are described by Brakenhoff et al. as silent point mutations. Therefore, the amino acid sequence of HGF, IFN-beta-2, 26kd protein and BSF-2 are considered to be different names for the same protein. For the purposes of the present invention, the amino acid sequence of mature, native, full-length IL-6 is considered to be the same as that of HGF, IFN-beta-2, 26kd protein, and BSF-2.
IL-6 has been reported to be an important cytokine with numerous significant biological activities. The activities of IL-6 often occur in conjunction with other growth factors, such as other interleukins and TNF. Thus, IL-6 is believed to play an important role in the regulation of inflammatory and immune responses to infection and injury. For example, IL-6 has been demonstrated to be involved in the proliferation and differentiation of B cells, T cells and multi-potential hematopoietic progenitor cells.
In addition, IL-6 has been observed to be involved in the inflammatory response and to induce various acute phase proteins in liver cells. Additional evidence of the involvement of IL-6 in the inflammatory response is the presence of high concentrations of IL-6 in the bodily fluids of patients with severe burns, kidney transplants, acute infections of the central nervous system, rheumatoid arthritis, and septic shock.
IL-6 has also been shown to stimulate megakaryocytopoiesis and platelet production. See McDonald et al., Blood 77, 735-740 (1991) and Hill et al., Blood 77, 42-48 (1991).
The biological activities of IL-6 suggest important immuno-therapeutic and anti-inflammation compositions. Immuno-therapeutic compositions containing IL-6 and other interleukins have, for example, been suggested by Kishimoto et al. (Ajinomoto Company, Inc.), European patent application 257,406.
Small amounts of human IL-6 may be isolated from natural sources, such as cells that secret IL-6. A number of cells secrete IL-6, including monocytes, T cells, fibroblasts, keratinocytes, and endothelial cells.
With the advent of recombinant DNA techniques, it has been found possible to produce larger amounts of pure IL-6 by expression in suitable host cells, such as E. coli, injected Xenopus oocytes, cell-free reticulocyte lysate, insect cells, and mammalian cells. For example, expression in Xenopus oocytes and cell-free reticulocyte lysate has been described by Revel et al. in UK patent application 2,063,882. Expression in E. coli has been reported by Snouwaert in the Journal of Immunology 146, 583-591 (1991); Brakenhoff et al. in the Journal of Immunology 145, 561-568 (1990), the Journal of Immunology 143, 1175-1182 (1989), and the Journal of Immunology 139, 4116-4121 (1987); Asagoe in Biotechnology 6, 806-809 (1988); and Yasueda in Biotechnology 8, 1036-1040 (1990).
Commercially, it is desirable to be able to produce proteins in E. coli. The ability to express large amounts of IL-6 in E. coli, however, has been reported to be associated with certain problems that need to be overcome. For example, Asagoe et al. were unsuccessful in expressing IL-6 in E. coli until they prepared an HGF fusion protein with a factor Xa-specific cleavage sequence; see Biotechnology 6, 806-809 (1988). Similarly, Yasueda et al. had to introduce dual Shine-Delgarno sequences in front of the coding region and employed A-T rich sequences between the Shine-Delgarno region and the initiation codon as well as in the codons for the N-terminal region of the protein in order to achieve high expression levels; see Biotechnology 8, 1036-1040 (1990).
In addition to being able to prepare large amounts of pure protein, recombinant DNA techniques permit molecular biologists to improve on the proteins that occur in nature. For example, it is possible to prepare proteins that lack some of the amino acids present in the native protein. Thus, Brakenhoff et al. has reported that the biological activity of IL-6 is not affected by deletion of up to 28 amino acid residues from the mature, native IL-6; see the Journal of Immunology 143, 1175-1182 (1989).
Native proteins may also be improved by substituting amino acids for one or more of the amino acids that occur naturally in a protein. Such substitutions may be introduced into a protein by expressing recombinant DNA having a nucleotide sequence modified so as to have a codon that represents the desired amino acid. The DNA may conveniently be modified using the technique of directed mutagenesis. Proteins expressed by such modified DNA are called muteins. Directed mutagenesis techniques have been reviewed by Lather et al. in Genetic Engineering, Academic Press, pages 31-50 (1983) and by Smith and Gillam in Genetic Engineering; Principles and Methods, Plenum Press, Volume 3, pages 1-32 (1981).
There have, for example, been recommendations to substitute cysteine residues in native interferon beta with other amino acids; see Mark et al., U.S. Pat. No. 4,853,332. The cysteine residues in interferon beta reportedly form undesirable inter-molecular and intramolecular bonds that affect activity and the ease with which the protein can be expressed in E. coli. Mark et al. speculate that the method may usefully be applied ". . . to any other biologically active protein that contains a functionally non-essential cysteine residue that makes the protein susceptible to undesirable disulfide bond formation."
Mark et al. recognized, of course, that one cannot always replace a cysteine residue and still retain biological activity. If the cysteine residue forms a disulfide bond that is essential to the tertiary structure of the protein, replacement of the cysteine will cause at least some loss of biological activity. According to Mark et al., the literature may be consulted for ". . . information regarding the cysteine content of biologically active proteins and the roles played by the cysteine residues with respect to activity and tertiary structure."
Recommendations have been made to replace one or more of the cysteine residues in IL-6; see Clark et al., PCT application WO88/00206. Several pieces of evidence, however, suggest that doing so is undesirable.
For example, one guideline suggested by Mark et al. in U.S. Pat. No. 4,853,332 for predicting which proteins are susceptible to having cysteine residues replaced by other amino acids is that such proteins usually have an odd number of cysteine residues. There are four cysteine residues in IL-6. Therefore, IL-6 is inconsistent with a guideline proposed by Mark et al.
In addition, Snouwaert et al., in the Journal of Immunology 146, 585-591 (1991), disclose that the replacement of the four cysteine residues in IL-6 resulted in significant loss of activity in four in vitro cell proliferation assays, especially when human cells were employed in the assays. This did not surprise Snouwaert et al., who noted that IL-6 and G-CSF are known to have significant homology, and that the disulfide structures of human IL-6 and human G-CSF are known to be similar; see Clogston et al., Archives of Biochemistry and Biophysics 272, 144-151 (1989). Since it is also known that substitution of any one of the four conserved cysteine residues in human G-CSF results in loss of biological activity, Snouwaert et al. reason that the loss of activity in cysteine-free IL-6 was expected.
Snouwaert et al. also conducted experiments to determine which regions of the IL-6 molecule are necessary for activity. They prepared a series of IL-6 mutants in which segments of twenty amino acids each were missing throughout the length of native IL-6. Only the mutant from which amino acid residues 4-23 were deleted retained significant activity. Each of the other deletions abolished activity. Snouwaert et al. conclude that disulfide bonding is important in maintaining the biologically active conformation of human IL-6.
Additional evidence for the importance of the cysteine residues for activity of IL-6 may be found in an article by Brakenhoff et al. in the Journal of Immunology 145, 561-568 (1990). Brakenhoff et al. used epitope mapping of specific monoclonal antibodies to study the active sites of IL-6. They conclude that there is an active site on the IL-6 molecule between amino acid residues 29 (glutamine) and 34 (leucine). This region is only 11 amino acids residues from the first cysteine residue at position 45. The proximity of the active site to this cysteine residue suggests that at least the first disulfide bond, and, therefore, the first two cysteine residues at positions 45 and 51, are necessary for activity. Brakenhoff et al. express the opinion that a second active site exists on IL-6, although the location of the second active site is uncertain.
Muteins and truncated versions of IL-6 are desirable. It is especially desirable, and the principal objective of the present invention, to produce cysteine-depleted muteins having at least comparable activity to that of native IL-6 despite evidence that such muteins do not exist.